This invention relates to an endometriosis mouse model wherein severely compromised immune deficient (SCID) female mice are transplanted with human xenografts of normal endometrial tissue, but result in mice with human endometriosis tissue. These diseased mice are useful in the study of endometriosis, in particular to identify nucleic acid sequences or amino acid sequences that up- or down- regulate the diseased state, or that are endometriosis specific.
Endometriosis is a disease affecting women of reproductive age, causing substantial debilitation, such as pelvic pain, and possible sterility or infertility, depending upon the severity of the condition. Most experts agree that endometriosis originates from retrograde menstruation of normal endometrial fragments that then implant on to peritoneal surfaces, from vascular or lymphatic dissemination of endometriosis lesions to other parts of the body, and/or from metaplasia, i.e., the abnormal transformation of one differentiated tissue into another. Modem Approaches to Endometriosis., eds. E. Thomas and J. Rocky, Kluwer Academic Publishers Boston (1991).
Typically, diagnosing endometriosis requires an invasive procedure, which results in the possibility of infection and other disadvantages associated with surgery. At present, the most effective therapy for treatment of endometriosis is surgical intervention, along with the administration of growth factor antagonists; ovarian suppression treatments, such as gonadotropin-releasing hormone (GnRH) agonists; and immunomodulators to inhibit the implantation of endometrial cells into undesired tissues. Therefore, it is desirable to develop procedures, techniques, and treatments that are easier to use and are more effective in the diagnosis and/or treatment of the disease.
One approach in general disease diagnosis has been to develop animal models that mimic certain disease states, and thereby study the indications of the disease and also study the impact of proposed treatment regimes. Primates are the only animal class to spontaneously develop endometriosis, although it is mainly a human disease condition. However, the use of non-human primates to study this disease is very expensive and labor-intensive due to the care required for the animals. Also, primates have a longer reproductive cycle than other laboratory animals, making a primate model less desirable as a system for preliminary endometriosis studies.
Human xenografts of normal endometrial tissue or endometriosis tissue have been transplanted into nude mice to create certain mouse models. See Zamah, et al., Transplantation of Normal and Ectopic Human Endometrial Tissue into Athymic Nude Mice , Am. J. Obstet. Gynecol., 149:591-597 (1984); Berqvist, et al., Human Uterine Endometrium and Endometriosis Tissue Transplanted into Nude Mice , Am. J. Pathol., 121:337-341 (1985); and Berqvist, et al., Human Endometrium Transplanted into Nude Mice , Am. J. Pathol., 119:336-344 (1985). More recently, SCID mice have been transplanted subcutaneously or intraperitoneally with normal human endometrium. See Aoki, et al., Successful Heterotransplantation of Human Endometrium in SCID Mice, Obstet.Gynecol., 83:220-228 (1994); and Awwad, et al., Immunohistochemical Characterization of Human Endometrial Transplants in SCID Mice Abstract P-315, page 236 (1995) presented at the Fifty-First Annual Meeting of the American Society for Reproductive Medicine.
A particular mouse model of surgically-induced endometriosis was evaluated to determine the effects of pregnancy on endometriosis and the effects of endometriosis on pregnancy. However, chopped mouse uterine tissue was utilized. See Cummings, et al., Effect of Surgically Induced Endometriosis on Pregnancy and Effect of Pregnancy and Lactation on Endometriosis in Mice , Endometriosis and Pregnancy in Mice, PSEBM, 212:332-337 (1996). The promotion of endometriosis using TCDD was discussed in Cummings, et al., Promotion of Endometriosis by 2,3,7,8-Tetrachlorodibenzo-p-dioxin in Rats and Mice: Time-Dose Dependence and Species Comparison, Toxicol. Appl. Pharmacol., 138:131-139 (1996).
However, the existing models result in the perpetuation and proliferation of the implanted material in its original form rather than exhibiting the progression of normal endometrial tissue to endometriosis diseased tissue, nor do these models indicate the progression of human tissue. Due to the relevance of the disease""s pathology, it is important to have a model system which mimics the development of the human disease state.
One aspect of the invention provides a novel mouse model for human endometriosis wherein normal human endometrial tissue grows and mimics the progression to human endometriosis. The endometriosis mouse model is prepared or generated by a method which comprises
a. eliminating endogenous progesterone from a severely compromised immune deficient (SCID) female mouse;
b. adding a micronized exogenous estrogen source to a xenograft of human normal endometrial tissue;
c. implanting the xenograft into the intraperitoneal cavity of the mouse;
d. adding an exogenous estrogen source to the mouse before and after implantation of the xenograft; and
e. allowing the xenograft to grow and mimic the progression of human endometriosis tissue. The length of time for progression is typically 3 to 7 weeks, preferably about 5 weeks.
Another preferred aspect of the invention is a female SCID mouse model for endometriosis, wherein said mouse is characterized by having a xenograft of human normal endometrical tissue implanted into the mouse""s intraperitoneal cavity and wherein the tissue is allowed to grow and progress to endometriosis.
Usually the endogenous progesterone is eliminated from the SCID mouse by a bilateral oophorectomy or by administration of an anti-progesterone agent. The mouse selected for the model is a female mouse in which her immune system is severely compromised, so that the implanted xenograft will be accepted, grow and develop.
In preparing the xenograft, a section of human normal endometrial tissue is identified, isolated from its human donor, and fragmented, and then an exogenous source of estrogen is added. Typically, this estrogen is micronized and is in water-soluble form prior to its administration or addition to the xenograft.
The prepared xenograft is implanted or transplanted into the intraperitoneal cavity of the mouse and exogenous estrogen is administered to the mouse both before and after implantation. The xenograft then grows and develops in the mouse, mimicking the progression of human endometriosis. Approximately from about 0.05 to about 1.5 cubic centimeters (cc) of the solid tissue is isolated with the xenograft optionally suspended in a physiologically-compatible solution or nutrient medium and the suspension injected into each mouse at an amount from about 0.4 cc to about 0.6, cc so that the mouse receives about 0.2 cc of material. Also, an antibiotic can be administered to the mouse in conjunction with the xenograft implantation.
The source of the xenograft tissue is from a human female; usually the result of a hysterectomy or an endometrial biopsy. Preferably, the xenograft is obtained from a pre-menopausal female not previously treated with gonadotropin-releasing hormone (GnRH) agonists.
Various sources of exogenous estrogen can be used, such as beta-estradiol-17-cypionate, poly-estradiol phosphate, beta-estradiol benzoate, and the like, and is administered to the xenograft and the mouse in different dosages and methods. The estrogen is added to the xenograft in a water-soluble or micronized form to reach a concentration from about 50 to about 500 nanomolars (nM); whereas the mouse receives from about 60 micrograms (ug) per kilogram (kg) per week to about 120 ug per kg per week of exogenous estrogen. The estrogen is administered intramuscularly or subcutaneously to the mouse starting at least one day prior to the implantation of the xenograft and continuing once a week after implantation.
This invention also provides molecular and immunohistochemical/immunopathological profiles in the mouse which are identical to human endometriosis, thus generating a model system to mimic the progression of normal endometrial tissue to diseased endometriosis tissue. Using the model, one of skill can screen and evaluate various potential therapies or prophylactics for treating or preventing endometriosis, since the model replicates the human clinical condition and provides clinical responses as an in vivo system. Further, the model provides a means to evaluate the effect of dosages, schedules, delivery systems, and routes of administration, as well as to identify nucleic acids or amino acids associated with the disease.